Ornamental cannabis sativa L. variety named &#39;Divina&#39;

ABSTRACT

The present invention provides a new ornamental variety of Cannabis sativa L., named ‘Divina’. ‘Divina’ was developed and obtained from a spontaneous mutation of the parent ‘Pilar’ and is asexually propagated, having a distinctive “mottled yellow and green leaf” phenotype.

CROSS REFERENCE TO RELATED APPLICATIONS

Not applicable.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT

Not applicable.

Latin name of the genus and species of the claimed plant: Cannabissativa L.

Variety denomination: ‘Divina’.

FIELD OF THE INVENTION

This invention relates to a new and distinct Cannabis ornamental plantwith the varietal denomination ‘Divina’, which is a variety of Cannabissativa L. and a spontaneous mutation of the varietal denomination‘Pilar’. ‘Divina’ is distinguished from the parent plant ‘Pilar’ by itsyellow and green variegated foliage as compared to the green foliage of‘Pilar’ as well as by other characteristics described below.

BACKGROUND OF THE INVENTION

The differences of opinion between taxonomists supporting monotypic andpolytypic concepts on Cannabis have not yet been resolved. However, themost accepted opinion is that Cannabis sativa L. is a highly variable,hybridized and introgressed, unstructured single plant species (Smalland Cronquist, 1976). This wide source of variation present in Cannabisgenetic resources has permitted breeders to select and develop varietieswith specific improved traits to be employed for obtaining medicinal,industrial and food derived products of added value. Breeding andcultivation of new Cannabis sativa L. varieties has recently increaseddue to the worldwide, growing demand in the fields of medicinal,industrial and food businesses. Hundreds of protected varieties arelisted in the International Union for the Protection of New Varieties ofPlants (UPOV) Plant Variety Database PLUTO, as evidenced when searchingfor protected varieties of Cannabis sativa L. (UPOV code: CANNB_SAT) inthe countries that signed the 1991 Act of the UPOV Convention.

The maximum content of the psychotropic compound Δ9-tetrahidrocannabinol(Δ9-THC) on a dry weight basis of the inflorescences in industrial andfood varieties is defined at a national level and ranges from 0.2% inEuropean Union, 0.3% in EEUU and up to 1.0% in countries such asSwitzerland, Uruguay and Colombia. In the last decade, medicinalvarieties with different chemotypes have been selected and a geneticmodel of inheritance has been completed and proposed by de Meijer andHammond (2016), resulting in an improved technical knowhow in breedingof medicinal varieties for producing a specific cannabinoid aspredominant, or in combination with other cannabinoids.

With the ongoing research and development of industrial and medicinalprotected varieties that does not possess contents of Δ9-THC higher than0.2-1.0%, the possibility of developing ornamental Cannabis varietiesmust be considered, especially when such protected varieties can beeasily distinguished from the rest of protected and unprotectedvarieties through the easy detection of the variegated foliage. Suchfoliage is a morphological marker that permits an easy and immediateidentification of specific protected Cannabis varieties commercializedas ornamentals.

Ornamental varieties account more than half of the total applications ofplant patents and plant breeder's rights (PBRs) granted in both EuropeanUnion and United States of America (Koo et al., 2004). Mohar Jain (2006)reported that within more than 2300 mutant varieties officially orcommercially released worldwide and registered at Joint FAO/IAEA MutantVariety Database, 566 represent ornamental varieties. Variegated plantscomprise approximately one-third of ornamental plants commercially grown(Gilman, 1996). Behe et al. (1999) reported that consumer preferencesfor plant characteristics have placed leaf variegation as second mostimportant characteristic to be considered in the purchase decision.

The cause of mutation (and in the case of ‘Divina’, the resulting leafvariegation) is mostly unknown. It can often appear spontaneously and iscalled “sporting.” Mutation may also be induced by a variety of factors,such as irradiation, chemical application or virus infection. Somemethods are extremely accessible and have been employed for centuries,while others involve advanced transgenic techniques listed by Frank andChitwood (2016). All these factors can change the genome, which in turnmay result in the inability to produce properly functional chlorophyll,and thus causing leaf variegation. Leaf variegation in higher plants haslong been known as a recessive genetic trait, and it results from adefect that makes chloroplast development unstable, since at least partof the tissues gives rise to normal chloroplasts. Leaf variegation wasproposed by Sakamoto (2003) to be a common phenotype of inter-organellarcompensation and cytoplasmic sorting processes that minimize the defectsin chloroplast and mitochondrial functions.

According to Kirk and Tilney-Basset (1978), variegation can becategorized as either cell lineage or non-cell lineage types. Celllineage variegation occurs in genetic mosaics (individuals with cells ofdifferent genotypes), while in non-cell lineage variegation all cellshave the same genotype but the genes responsible for the trait orphenotype are expressed only in some of the cells, e.g., because ofdifferential gene expression (Marcotrigiano, 1997).

Leaf variegations can arise from mutations that make chloroplastformation unstable; while these mutations are most often found in thechloroplast genome they can also occur in the nuclear genome (Sakamoto,2003). Mutations in the nuclear genome can be distinguished frommutations in the chloroplast genome by the inheritance pattern. Morespecifically, chloroplast mutations are typically inherited maternally,while mutations in the nuclear genome show Mendelian inheritance(Fawole, 2001).

Cannabis genetic resources are highly variable in their aestheticappearance and in morphological, physiological and biochemicalcharacteristics of relevance. Cannabis plants grow quickly andvigorously under optimal growing environments, being generallyappreciated as attractive-looking ornamental plant species for theabundant green foliage, the characteristic leaf and the aromaticinflorescences. Because of the legal restrictions on cultivatingindividual plants, the ornamental industry has never considered thisplant species as an ornamental crop. Only the variety ‘Panorama’ wascommercialized as ornamental in Hungary in the 1980s, being bred by IvánBócsa for its short and strongly-branched growth habit (Small andMarcus, 2002). The limited success of this ornamental variety was mainlycaused by the fact that it was not easily distinguishable from the restof protected, and unprotected Cannabis varieties, thereby potentiallyassociating its cultivation as an illegal act.

By contrast, and despite of the potential risk of poisoning for smallchildren and domestic livestock, beautiful and attractive ornamentalvarieties of toxic, psychotropic or medicinal plant species are widelycultivated in private and public areas, such as for example: pinkoleander (Nerium oleander), yellow oleander (Thevetia peruviana),poinsettia (Euphorbia pulcherrima), hydrangea (Hydrangea macrophylla),rhododendron (Rhododendron ponticum), Saint Peter cactus (Echinopsispachanoi), wormwood (Artemisia absinthium), opium poppy (Papaversomniferum), foxglove (Digitalis purpurea), periwinkle (Catharanthusroseus), etc.

In April 2017, we obtained provisional protection obtained in plantbreeder rights (PBR) from the Community Plant Variety Office (CPVO) forthe dioecious female ornamental variety ‘Divina’ (CPVO file number:2017/0149). During the pendency of the PBR applications, ‘Divina’ wasnot publicly available as testing sites for DUS (Distinctness,Uniformity, Stability) trials, such as Naktuinbouw in The Netherlands,are under the control of the CPVO. The PBR application for ‘Divina’ wasoriginally submitted to the CPVO under the proposed demonination name‘Enza’ and subsequently changed to ‘Divina’. As of the date of filing ofthis plant patent application, ‘Divina’ has not been publicly availableor offered for sale anywhere in the world nor has it been offered forsale under another variety name.

‘Divina’ is one of the first Cannabis varieties to obtain protection ofplant breeder's rights at the CPVO, granted with decision N° EU 50045 of16 Jul. 2018. ‘Divina’ is characterized by a stable variegated foliageof the vegetatively-derived propagated plants. ‘Divina’ has adistinctive “mottled yellow and green leaf” phenotype as compared to itsparent, the medicinal variety ‘Pilar’ (CPVO Application number2016/0115; granted with decision N° EU 50009 of 16 Jul. 2018). ‘Divina’was detected from a spontaneous bud sport of ‘Pilar’. Both ‘Divina’ and‘Pilar’ are characterized by having cannabidiol (CBD) as predominantcannabinoid, instead of Δ9-THC. The distinctness easily detected byusing a variegated foliage as morphological marker (FIGS. 1-3) is animpetus in the commercialization of ornamental Cannabis varieties suchas ‘Divina’.

We were interested in performing a direct comparison between DNAfingerprinting of the mutated variety ‘Divina’ and the parent variety‘Pilar’. We used a set of 15 Simple Sequence Repeat (SSR) markers usedfor ensuring genetic authenticity and traceability, and for enforcingPBRs of protected varieties at our company. Besides the interest indetecting differences at genotypical and phenotypical levels, we werealso interested in detecting potential differences at microscopic andchemotypical levels. For this reason, a molecular marker for Δ9-THCA andCBDA synthases, gas chromatography, conventional and advanced microscopywere used as tools to research on the distinct morphological andphysiological characteristics of genetically distinct cells, the “greencell” and the “yellow cell”. An objective of this study was to detectdifferences and to evaluate the correlations between the mutated variety‘Divina’ and the un-mutated parent variety ‘Pilar’.

Plants of the variety ‘Divina’ have a significant lower content ofchlorophyll in their leaves than plants of the variety ‘Pilar’. A slowergrowth rate and less total biomass production have been generallyobserved when mother plants of both varieties are grown at similarconditions under long daylength, supposedly due to the inability of‘Divina’ to synthesize chlorophyll in portions of the photosyntheticorgans. The main objectives of the second example described below wereto evaluate yield losses or gains in vegetal raw material components(stalks, leaves and inflorescences) between the mutated variety ‘Divina’and the parent variety ‘Pilar’, as well as cannabinoid and terpenoidyields. A comparative production trial between plants of the mutatedvariety ‘Divina’ and plants of the un-mutated variety ‘Pilar’ was set inthe same trial location for evaluating yield losses or gains in vegetalraw material components (stalks, leaves and inflorescences). Cannabinoidand terpenoid yields and possible changes in specific secondarymetabolites due to genomic mutation were also examined. Our studiesshowed differences in CBD and Δ9-THC amounts in ‘Pilar’ vs. ‘Divina’.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a new and distinct ornamental variety ofCannabis sativa L. with a varietal denomination of ‘Divina’.‘Divina’ wasidentified as a spontaneous mutation of the varietal denomination‘Pilar’. The variety ‘Divina’ has a distinctive “mottled yellow andgreen leaf” phenotype as compared to the green-leafed parent variety‘Pilar’. ‘Divina’ also differs from the parent plant ‘Pilar’ in having alower amount of chlorophyll (about 73% of ‘Pilar’), slower growth andlower height, less biomass, and lower CBD content (less than 4%) andlower Δ9-THC content (less than 0.4%) in inflorescences than ‘Pilar’.

‘Divina’ obtained a certificate of plant breeder's rights at the CPVO,granted with decision N° EU 50045 of 16 Jul. 2018.

The mutated plant ‘Divina’ was detected and isolated from a bud sport ofthe donor plant (‘Pilar’) growing in greenhouse. The first stablevariegated clone, used as the mother plant for future generations, wasobtained after seven generations of asexual propagation of the originalisolated chimera. Approximately 12-13 generations have been tested forstability. Asexual propagation is commonly performed by in vivo rootedcuttings of medicinal varieties (Potter, 2013), but also effective invitro micropropagation protocols have been developed (Lata et al.,2017). Elite stock plants are maintained in repositories in vegetativestage under artificially created long daylength controlled environmentfor generating propagules for in vivo or in vitro propagation. Aprotocol for all the stages of in vitro micropropagation is alreadyestablished at our laboratories. Propagation of true-to-type variegated‘Divina’ rooted cuttings can be performed satisfactorily by adoptinggood in vivo and in vitro propagation practices and the clones areidentical to the original ‘Divina’ in all distinguishingcharacteristics. Cuttings from ‘Divina’ have demonstrated that thecombination of characteristics disclosed herein are stable and firmlyfixed, and are retained true-to-type.

When considering the sexual propagation of the variety, all theF1-hybrid offspring obtained by crossing a dioecious “green leaf” maleplant with a ‘Divina’ plant show a “green leaf” phenotype.

Both ‘Divina’ and ‘Pilar’ varieties show the same genotype when DNAfingerprinting is performed by using 15 SSR molecular markers.Additionally, by using a molecular marker for Δ9-THCA/CBDA synthases itwas confirmed that only the alleles for CBDA synthase can be detected inboth varieties, as it is also confirmed by using gas chromatography thatboth varieties have a CBD chemotype.

The content of the psychotropic compound Δ9-THC in inflorescences of‘Divina’ plants was lower than 0.4% in all the samples analyzed at ourlaboratories. The content of CBD was less than 4% in inflorescences of‘Divina’ plants (Codesido et al., 2018). Microscope observations of“yellow” portions of stalks, leaves and inflorescence bracts confirm thepresence of bulbous, sessile- and capitate-stalked trichomes.

‘Divina’ cuttings are more prone to fungal disease during in vivorooting stage than ‘Pilar’ cuttings, however satisfactory percentages oftrue to type rooted cuttings can be achieved by adopting goodpropagation practices. In artificial growing conditions the variety‘Pilar’ achieved significantly higher cannabinoids and terpenoids yieldsthan the variety ‘Divina’. Slight differences of terpenoid compositionwere also detected.

Plants of the present invention have not been observed under allpossible environmental and cultural conditions. The phenotype may varysomewhat with variations in environmental conditions, especially whenplants are exposed to completely natural conditions, without, howeverany variance in the genotype. For example, phenotypic expression mayvary somewhat with fluctuations in temperature and CO2, light spectraand intensity, substrate volume and chemistry, for mentioning the mostinfluential parameters.

BRIEF DESCRIPTION OF THE PHOTOGRAPHS

The accompanying colored photographs illustrate the overall appearanceof ‘Divina’ showing the colors as true as it is reasonably possible toobtain in colored reproductions of conventional photography. Thephotographs were taken in a greenhouse setting under defused, naturallighting.

FIG. 1A-B: FIGS. 1A and 2B show a close up view of the yellow and greenvariegated foliage of the ‘Divina’ variety. The photograph is of a stockplant of approximately six weeks of age maintained under 18 hours/day ofartificial lighting (400-600 μmol m⁻²s⁻¹), under controlled temperaturesand relative humidity, and CO₂ enriched up to 800 ppm.

FIG. 2: FIG. 2 shows individual plants of the ‘Pilar’ (left) and‘Divina’ (right) varieties for purposes of comparison of leaf color,variegation, and size. The parent plant ‘Pilar’ has green foliage whilethe spontaneous mutant ‘Divina’ has variegated yellow and green leaves.Under the same growth conditions, ‘Divina’ is shorter than ‘Pilar’. Thephotograph is of stock plants of approximately six weeks of agemaintained under 18 hours/day of artificial lighting (400-600 μmolm⁻²s⁻¹), under controlled temperatures and relative humidity, and CO₂enriched up to 800 ppm.

FIG. 3: FIG. 3 shows a close up view of a ‘Divina’ flower and attachedleaves of an auxillary branch. The photograph is of a flowering plant often weeks of age; four weeks of vegetative stage and six weeks ofreproductive stage.

EXAMPLES Example 1: Development of an Ornamental Cannabis sativa L.Variety: Phytochemical, Morphological, Genetic Characterization andPropagation Aspects MATERIALS AND METHODS

The spontaneously mutated type chimera was detected on 25^(th) of May2015 and isolated from a mutated donor plant growing in greenhouse. Onlyone cutting was isolated and rooted from the first generation of thechimera and, after various generations of vegetative propagation ofselected cuttings developing a partially variegated leaf foliage inderived mother plants, leaf variegation in all the leaves of selectedmother plants was achieved starting from the seventh generation of thechimera. Some other not selected mother plants at this generation werestill expressing some individual “not mottled green leaf” and/or “notmottled yellow leaf”, and even “not mottled green branch” and/or “notmottled yellow branch.” Other chimeras have been detected in differentmother plants of the variety ‘Pilar’, suggesting its high propensity todevelop spontaneous mutations.

DNA of three leaf samples taken from elite stock plants maintained inthe repository were extracted by using the DNeasy Plant Mini Kit (CatNo./ID: 69104) from Qiagen. DNA samples were identified as: “Pilar notmottled green leaf”, “Divina variegated leaf”, and “Divina not mottledyellow leaf” and sent to the Center for Research in AgriculturalGenomics (CRAG), which is a public consortium formed by various Spanishinstitutions (CSIC, IRTA, UAB and UB) and placed in Barcelona. The setof 15 SSRs and the molecular marker for Δ9-THCA/CBDA synthases weredeveloped and carried out by researchers at CRAG. After confirming thegood quality of the received DNA samples, PCR amplification of 15 SSRmarkers labelled with fluorescence was performed, followed by separationand detection of SSRs using an automatic sequencer of type ABI Prism®3130xl (Applied Biosystems). Genetic analysis was performed withsoftware GeneMapper® Version 4.0 (Applied Biosystems). The molecularmarker for Δ9-THCA/CBDA synthases and 15 molecular markers are notpublicly disclosed, being these identified with increasing numbers fromCAN1 to CAN15.

Two different microscopic techniques were employed for detectingextracellular and intracellular variability between leaf samples (in theform of discs with 1 cm of diameter) identified as “Pilar not mottledgreen leaf” and “Divina not mottled yellow leaf”. A conventionalmicroscopy approach by using the binocular microscope BA310 from Moticwas employed to visually detect and confirm the presence of glandulartrichomes in the upper and lower surface of leaf discs, as well as in“yellow stalks” and “yellow inflorescences bracts”. An advancedmicroscopy approach by using a transmission electron microscopy JEOLJEM-1400 (TEM) from SEMTech Solutions was employed to study thedifferences in cell organelles of leaf discs and to take images. Leafdiscs for TEM visualization were stored in glutaraldehyde solution untilwere sent to the Servicio Central de Apoyo a la Investigación (SCAI) ofthe University of Córdoba, where researchers prepared samples bydehydrating, fixing and cutting them into very thin cross-sections byusing the ultramicrotome ULTRACUT R from Leica for preparing microscopegrids. TEM images of cell organelles were obtained and analyzed incollaboration with a specialist in Anatomy and Pathological AnatomyCompared.

A comparative rooting trial was performed by taking 180 cuttings fromelite stock plants of each variety maintained in the repository atPhytoplant Research S.L. The basal part of the cutting was immersed in aliquid rooting product (Inabar) containing natural phytohormones, andthen treated cuttings were placed in Jiffy cubes (model 7; 44 mm)disposed in propagation greenhouses where 60 treated cuttings can beplaced in each unit during the in vivo rooting stage. The range adoptedfor climatic parameters were: 22±1° C., 75-85% of relative humidity,350-400 ppm of carbon dioxide, and under a photon flux density ofapproximately 100 μE mol m⁻²s⁻¹ provided by light-emitting diodes (LEDs)lights under continuous daylength (24 hours light/day). After threeweeks from taking cuttings, data were taken for dead cuttings fromfungal disease and for healthy rooted cuttings.

At the same time, a generic protocol for all the stages of in vitromicropropagation already established at Phytoplant Research S.L.laboratories for axillary shoots of the variety ‘Pilar’ was also testedfor performing the in vitro micropropagation shoots of the variety‘Divina’. Shoots were incubated in a growth chamber (model A1000 fromConviron) and the range adopted for climatic parameters were: 25±1° C.,70-80% of relative humidity, 350-400 ppm of carbon dioxide, and underphoton flux density of approximately 225 μE mol m⁻²s⁻¹ provided byfluorescent tubes under long daylength (18 hours light/day). A batch ofF1-hybrid seeds was produced by using a dioecious “green leaf” maleselected plant (“79/4/1/5/1”) as a male parental plant. 73 F1-hybridseeds of the cross “79/4/1/5/1” X ‘Divina’ were germinated andcultivated during a period of 14 weeks under artificially created longdaylength controlled environment for evaluating the expression ofvariegated leaves in plants of the F1-hybrid offspring.

RESULTS AND DISCUSSION

By using the molecular marker system developed at CRAG we have beenpreviously able to distinguish several Cannabis genetic resourcesincluded in CRAG database, ranging from unprotected to protectedvarieties, both sexually propagated monoecious and dioecious varietiesas well as asexually propagated dioecious female varieties (data notshown). In Tables 1, 2, 3 are shown the alleles detected (in base pairs)with the 15 SSR markers. Both varieties ‘Divina’ (identified as “Divinavariegated leaf”) and ‘Pilar’ (identified as “Pilar not mottled greenleaf”) shown the same genotype when DNA fingerprinting was performed byusing 15 SSR markers, as well as when analyzing a mutant “yellow leaf”phenotype sample (identified as “Divina not mottled yellow leaf”). Thelack of genetic differences in base pairs of the alleles detected,especially between samples of “Pilar not mottled green leaf” and “Divinanot mottled yellow leaf”, evidenced that the spontaneous geneticmutation resulting in plant tissues devoid of chlorophyll cannot bedetected by the 15 SSR markers. The SSRs CAN2, CAN5, CAN7, CAN12a andCAN14 were homozygous in the three samples ‘Divina’, “Divina not mottledyellow leaf” and ‘Pilar’ (see Tables 1, 2, 3).

Tables 1-3. Alleles detected (in base pairs) with molecular markersidentified from CAN1 to CAN5 (Table 1), from CAN6 to CAN10 (Table 2),from CAN11 to CAN15 (Table 3). The abbreviation “Divina yellow” is usedfor samples of “Divina not mottled yellow leaf”.

TABLE 1 SAMPLE ID CAN CAN2 CAN3 CAN4 CANS ‘Divina’ 121 130 162 162 213225 282 287 115 115 “Divina_yellow” 121 130 162 162 213 225 282 287 115115 ‘Pilar’ 121 130 162 162 213 225 282 287 115 115

TABLE 2 SAMPLE ID CAN6 CAN7 CAN8 CAN9 CAN10 ‘Divina’ 165 167 214 214 267269 158 175 185 188 “Divina_yellow” 165 167 214 214 267 269 158 175 185188 ‘Pilar’ 165 167 214 214 267 269 158 175 185 188

TABLE 3 SAMPLE ID CAN11 CAN12a CAN13 CAN14 CAN15 ‘Divina’ 208 223 287287 280 288 260 260 160 164 “Divina_yellow” 208 223 287 287 280 288 260260 160 164 ‘Pilar’ 208 223 287 287 280 288 260 260 160 164

Additionally, by using a molecular marker for Δ9-THCA/CBDA synthases itwas confirmed that only the alleles for CBDA synthase can be detected inboth varieties, as it was confirmed by using gas chromatography thatboth varieties have a CBD rich-chemotype. In fact, in Table 4 are shownthe alleles detected at the B locus, known for determining Cannabischemical phenotype. A model involving one locus, B, with two alleles,B(D) and B(T), have been proposed (de Meijer et al., 2003), with the twoalleles being codominant. However, recently Weiblen et al. (2015)suggested that Δ9-THCA and CBDA synthases are possibly encoded by morethan one locus and that these loci are genetically linked. Pure CBDchemotype plants are supposedly due to homozygosity at the B locus andonly the alleles B(D)/B(D) were detected and expressed as CBDA synthase.The content of Δ9-THC in inflorescences of ‘Divina’ plants was lowerthan 0.4% in all the samples analyzed at our laboratories by gaschromatography, while CBD content was lower than 4% (Codesido et al.,2018).

TABLE 4 Alleles detected [B(D) or B(T)] at B locus with a molecularmarker for Δ9-THCA/CBDA synthases. Δ9-THCA/CBDA SAMPLE ID synthases“Divina variegated leaf” B(D) B(D) “Divina not mottled yellow leaf” B(D)B(D) “Pilar not mottled green leaf” B(D) B(D)

Binocular microscope observations of “yellow” portions of stalks, leavesand inflorescence bracts of ‘Divina’ variety confirmed the presence ofbulbous, sessile- and capitate-stalked trichomes in “yellow” portions ofstalks and inflorescence bracts, as well as in both upper and lowersurface of leaf discs, with a higher concentration of glandulartrichomes in the upper surface. In some samples of leaf discs of‘Divina’, especially in the “yellow” portions of upper surface of leafdiscs, we have observed anthocyanins accumulation. In other observationson plants of ‘Divina’ and ‘Pilar’ cultivated under differentartificially created long daylength controlled environments, we havedetected that under certain undisclosed conditions the accumulation ofanthocyanins can be visually observed in ‘Divina’ apical leaves directlyexposed to artificial light, while cannot be observed in ‘Pilar’.

Transmission electron microscopy observations on leaf discs permitted usto detect evident differences in cell organelles from the twogenetically distinct cells, “green cell” and “yellow cell”. In “yellowcell” highly vacuolated leucoplasts were detected; however, nucleiexhibit usual shape as in “green cell”, meaning that they are normallyperforming the function of governing gene expression and facilitatingDNA replication during the cell cycle. Leucoplasts are involved inmonoterpene biosynthesis and several secretory cells contain trueleucoplasts, devoid of thylakoids and ribosomes (Cheniclet and Carde,1985). However, in leucoplasts of “yellow cell” thylakoids were detectedas well as granules. Those granules were also identified in “green cell”and could be tentatively considered as terpenic granules, whichsupposedly discharge their content to the vacuole; however, it is stillnot known how occur the mechanism of transportation of terpenes from oneregion to another region within the plant cell or to outside the plantbody.

The results of the comparative rooting trial have shown significantdifferences at the 0.05 level when one-population t-Tests were performedon obtained data, except for the % of not rooted cuttings. ‘Divina’cuttings were significantly (p=001874) more prone (21.7%) to fungaldisease during in vivo rooting stage than ‘Pilar’ cuttings (3.3%), beingparticularly susceptible those cuttings having a less lignified stalk. Asignificantly (p=0.00844) higher % of healthy rooted cuttings wasachieved with ‘Pilar’ cuttings (92.2%), however satisfactory percentagescan be also achieved with ‘Divina’ cuttings (70.6%). A protocol for allthe stages of in vitro micropropagation was already established at ourlaboratories, and it was the same protocol generally adopted for shoottips of the variety ‘Pilar’. After the surface-sterilization of axillaryshoot tips, they were introduced in vitro where they generally lastednine weeks, and then additional two weeks for ex vitro hardening beforebeing used as propagation material.

Three steps were performed in vitro by using the same medium compositionbut by changing volume and type of containers: 1. induction of shoots (1mL in multi-well plates for 1 week), elongation of shoots (5 mL in deWit tubes for 4 weeks), and rooting (50 mL in Eco2Box oval containersfor 4 weeks). The medium used was: Formula β (Casano and Grassi,2009)+3% (w/v) sucrose at the step 1 and 1.5% (w/v) sucrose during steps2 and 3+0.8% (w/v) agar+2.0 μM meta-Topolin (Lata et al., 2016), atpH=5.8.

When considering the sexual propagation of the variety, all theF1-hybrid offspring composed of 73 plants obtained by crossing adioecious “green leaf” male plant with a ‘Divina’ plant shown a “greenleaf” phenotype, confirming that leaf variegation is a recessive genetictrait, and that voluntary or involuntary pollination of ‘Divina’ femaleinflorescences should not be used for the transmission of the trait leafvariegation in directly derived F1-hybrid offspring.

CONCLUSIONS

Although no differences were detected at genotypical level between‘Divina’ and ‘Pilar’ varieties by adopting specific molecular markers,evident differences can be detected at phenotypical level, especiallywhen “green cell” and “yellow cell” are examined more in details. Nextgeneration sequencing technologies need to be applied in both varietiesfor discovering exactly where the mutation occurs, if in the nuclear orchloroplast genome. Further cytological, chemical, genetic, andmolecular research need to be performed to study the biological natureof the mutated type chimeras, and to unveil the genetic inheritance andsexual transmission of the trait leaf variegation to other Cannabisgenetic resources. Propagation of true to type variegated ‘Divina’rooted cuttings can be performed satisfactorily by adopting good in vivoand in vitro propagation practices.

LITERATURE CITED

-   Behe, B., Nelson, R., Barton, S., Hall, C., Safley, C. D., and    Turner, S. (1999). Consumer Preferences for Geranium Flower Color,    Leaf Variegation, and Price. HortScience 34, 740-742.-   Casano, S., and Grassi, G. (2009). Valutazione di terreni di coltura    per la propagazione in vitrodella canapa (Cannabis sativa L.).    Italus Hortus 16, 109-112.-   Casano, S. (2018). Development of ornamental Cannabis sativa L.    varieties: phytochemical, morphological, genetic characterization    and propagation aspects. Acta Horticulturae (under editor review).-   Cheniclet, C., and Carde, J. P. (1985). Presence of leucoplasts in    secretory cells and of monoterpenes in the essential oil: a    correlative study. Journal of Botany 34, 219-238.-   Codesido, V., Marin, M., Sánchez-Carnerero Callado, C.,    Ferreiro-Vera, C., and Casano, S. (2018). Cannabinoids and    terpenoids yields of the ornamental Cannabis sativa L. variety    Divina characterized by a variegated foliage as morphological    marker. Acta Horticulturae (under editor review).de Meijer, E. P.    M., Bagatta, M., Carboni, A., Crucitti, P., Moliterni, V. M.,    Ranalli, P., and Mandolino, G. (2003). The inheritance of chemical    phenotype in Cannabis sativa L. Genetics 163, 335-346. PubMed-   de Meijer, E. P. M., and Hammond, K. M. (2016). The inheritance of    chemical phenotype in Cannabis sativa L. (V): regulation of the    propyl/pentyl cannabinoid ratio, completion of a genetic model.    Euphytica 210, 291-307 https://doi.org/10.1007/s10681-016-1721-3.    PubMed-   Fawole, I. (2001). Maternal inheritance of plant variegation in    cowpea, Vigna unguiculata (L.) Walp. Theoretical and Applied    Genetics 102, 459-462 https://doi.org/10.1007/s001220051667. PubMed-   Frank, M. H., and Chitwood, D. H. (2016). Plant chimeras: The good,    the bad, and the ‘Bizzaria’. Developmental Biology 419, 41-53    https://doi.org/10.1016/j.ydbio.2016.07.003. PubMed-   Gilman, E. F. (1996). Betrock's Florida Plant Guide. Betrock    Information Systems, Hollywood, Fla.-   Kirk, J. T. O., and Tilney-Bassett, R. A. E. (1978). The plastids:    Their chemistry, structure, growth and inheritance, 2^(nd) eds    (Amsterdam, N.Y. and Oxford: Elsevier/North-Holland Biomedical    Press), pp.960.-   Koo, B., Nottenburg, C., and Pardey, P. G. (2004). Plants and    Intellectual Property: An International Appraisal. Science 306,    1295-1297 https://doi.org/10.1126/science.1106760. PubMed-   Lata, H., Chandra, S., Techenalkhlas, N., Mahmoud, K., and El    Sohly, A. (2016). In vitro mass propagation of Cannabis sativa L.: A    protocol refinement using novel aromatic cytokinin metatopolin and    the assessment of eco-physiological, biochemical and genetic    fidelity of micropropagated plants. J. of App. Res. on Medicinal and    Aromatic Plants 3, 18-26    https://doi.org/10.1016/j.jarmap.2015.12.001. PubMed-   Marcotrigiano, M. (1997). Chimeras and variegation: patterns of    deceit. HortScience 32, 773-784.-   Mohan Jain, S. (2006). Mutation-assisted breeding for improving    ornamental plants. Acta Hortic. 714, 85-98    https://doi.org/10.17660/ActaHortic.2006.714.10. PubMed-   PLUTO (UPOV Plant Variety Database). (2018).    http://www.upov.int/pluto.-   Sakamoto, W. (2003). Leaf-variegated mutations and their responsible    genes in Arabidopsis thaliana. Genes and Genetic Systems 78, 1-9    https://doi.org/10.1266/ggs.78.1. PubMed-   Small, E., and Marcus, D. (2002). Hemp: A new crop with new uses for    North America. In Trends in new crops and new uses. J. Janick and A.    Whipkey eds (Alexandria, Va.: ASHS Press), p.284-326.-   Weiblen G. D., Wenger, J. P., Craft, K. J., ElSohly, M. A.,    Mehmedic, Z., Treiber, E. L., and Marks, M. D. (2015) Gene    duplication and divergence affecting drug content in Cannabis    sativa. New Phytologist 208, 1241-1250

Example 2: Cannabinoid and Terpenoid Yields of the Ornamental Cannabissativa L. Variety ‘Divina’ Characterized by a Variegated Foliage asMorphological Marker MATERIALS AND METHODS

The method described by Lichtenthaler (1987) for extracting andquantifying chlorophyll in μg/mL was employed to measure chlorophyll in‘Pilar’ (not mottled green leaves) and in ‘Divina’ (variegated leaves)from 6 samples of each variety taken from representative leaves directlyexposed to artificial light. Sampled mother plants were cultivated underlong daylength and used as sources of propagules for the in vivo rootedcuttings used in the trial.

The comparative production trial was performed at Phytoplant Researchauthorized facilities in Córdoba (Spain). A cabinet for indoorcultivation of 2 m² of cultivated area. A total of eight plants for eachvariety (8 plants/m²) were cultivated. The cabinet was equipped with two600 W/m² Philips Greenpower 600 W 400V EL lights providing a photon fluxdensity of approximately 700 μmol m⁻²s⁻¹. Plants were cultivated in 8.5L pots filled and maintained for 4 weeks in vegetative stage under longdaylength (18 hours light/day) and then induced to flower under shortdaylength (12 hours light/day) and maintained for additional 5 weeks,when plants were harvested and data on individual plant height and totalfresh weight (FW) were taken. The range adopted for climatic parameterswere: 25±3° C., 50-70% of relative humidity, and 350-400 ppm of carbondioxide. Plants were cultivated on a coconut natural growth medium (CocoProfessional Plus, Canna Continental, Los Angeles, Calif.) and nutrientsolutions were prepared by using the fertilizers Coco A+B from CannaContinental. Nutrient solution was applied to plants at variable valuesof electrical conductivity during the various phenological stages (from1.2 to 1.8 mS/cm; 0.4 mS/cm during the last week of cultivation).

After the drying stage, data on total dry weight (TDW) were obtainedafter manual processing, separation and taking the respective data ondry weights of vegetal raw material components: leaves (LDW),inflorescences (FDW), and stalks (SDW). Approximately five grams ofinflorescences and five grams of leaves were sampled for each one of the16 plants. Samples were differently prepared, stored and extractedbefore cannabinoids and terpenoids analysis, depending from thesecondary metabolites to be analyzed.

For cannabinoid analysis an Agilent 7890 B gas chromatograph was used.Separation of compounds was performed by using a fused silica capillarycolumn Rxi-35Sil_MS (15 m×0.25 mm I.D., 0.25 μm thickness of the film).Gas carrier was helium (99.9%) with a flow of 2.5 mL/min. The program ofthe oven was as follows: 200° C. as starting temperature, maintainedduring 0.1 min, increasing 120° C./min up to 250° C. maintained during 5min and 50° C./min up to 300° C. Injection of 1 μL at 250° C. inSplitless. Compounds were detected by mass spectrometry using a MS 5977Bfrom Agilent. The transfer line was maintained at 330° C., the source ofionization at 230° C. and the quadrupole at 150° C. Data acquisition wasby SCAN mode, with an interval of m/z between 40 and 400.

For terpenoid analysis, an Agilent 7820 B gas chromatograph was used.Separation of compounds was performed by using a fused silica capillarycolumn HP-5MS UI (30 m×0.25 mm I.D., 0.25 μm thickness of the film). Gascarrier was helium (99.9%) with a flow of 1.5 mL/min. The program of theoven was as follows: 50° C. as starting temperature, maintained during0.1 min, increasing 4° C./min up to 70° C. maintained during 14 min, 6°C./min up to 77° C. maintained during 9.5 min, 50° C./min up to 165° C.,1° C./min up to 170° C. and 25° C./min up to 300° C., maintained during5 min. Injection at 220° C. in splitless of 1 μL. The detector wasmaintained at 300° C. by employing 35 mL/min of H2, 350 mL/min of airand 25 mL/min of N₂, registering the signal of instrument at 20 Hz.

The data were subjected to t-Student Test to evaluate the significanceof differences on means of the different media by using MicroCal Originversion 4.1 statistical software package.

RESULTS AND DISCUSSION

Results obtained on chlorophyll contents by using the destructive methoddescribed by Lichtenthaler (1987) have confirmed that plants of thevariety ‘Divina’ had a significant lower content of chlorophyll thanplants of the variety ‘Pilar’, being 10.97 μg/mL the average chlorophyllcontent of the leaves of the variety ‘Divina’, representing 73% of theaverage detected in leaves of the variety ‘Pilar’, which was 15.02 μg/mL(Table 1). These results confirmed the observations that plants of thevariety ‘Divina’ had generally a slower growth rate and less totalbiomass production when mother plants of both varieties were grown atsimilar conditions under long daylength, supposedly due to the inabilityto synthesize chlorophyll in portions of the photosynthetic organs(Codesido et al., 1980).

TABLE 1 Chlorophyll contents in different samples of ‘Divina’ and‘Pilar’ varieties. Clorophyll content SAMPLE ID (μg/mL) SD SE Sum N‘Divina’ 10.97 1.51 0.61 65.79 6 ‘Pilar’ 15.02** 2.97 1.21 90.12 6**mean significant statistical differences by using the t-student test;p < 0.05

Significant differences at the 0.05 level were shown when one-populationt-Student Test was performed on some of the obtained data. Dataconfirmed previous observations on mother plants of both varieties grownat similar conditions under long daylength, and shown that in artificialgrowing conditions flowered plants of the variety ‘Pilar’ achieved asignificantly higher height (p=9.36E⁻⁵) and SDW (p=0.046) than floweredplants of the variety ‘Divina’, confirming that a deprivedphotosynthetic activity result in less total biomass accumulation in thestalks, the only vegetal raw material component not of interest forobtaining Cannabis-based medicines. In fact, no significant differenceswere found on FW, TDW, LDW and FDW, confirming that similar yield of thevegetal raw material components (leaves and inflorescences) used forextracting cannabinoids and terpenoids can be obtained from bothvarieties, although slightly higher yields on LDW and FDW were obtainedfrom plants of the variety ‘Pilar’.

Significant differences at the 0.05 level were shown when one-populationt-Student tests were performed on most of the obtained gaschromatographic data on CBD, Δ9-THC, and total terpenoids contents inleaves and inflorescences. Concentrations of most analytes weresignificantly higher in samples of ‘Pilar’ variety. No significantdifferences were found on Δ9-THC content in leaves (p=0.05) and totalterpenoids content in inflorescences (p=0.69). Average contents of CBDwere significantly lower in inflorescences (3.22%) and in leaves (1.57%)in plants of the variety ‘Divina’ (respectively p=2.16E⁻⁶ andp=0.00883). In a similar way, average contents of the psychotropiccompound Δ9-THC were significantly lower in inflorescences (0.34%) andin leaves (0.15%) in plants of the variety ‘Divina’ confirming that thepotential risk of abuse is inferior than with plants of the variety‘Pilar’. Average contents of total terpenoids in plants of the variety‘Divina’ were 0.80% in inflorescences and 0.34% in leaves, with averagecontents of total terpenoids in inflorescences not being significantlydifferent between plants of the different varieties.

The results obtained on cannabinoid contents of ‘Divina’ variety are notcompletely in agreement with the results from a previous study (Potter,2009) performed on an unprotected Cannabis variety with variegatedportions of foliage. In fact, Potter (2009) reported that leaf tissueslacking chlorophyll suffered no reduction in cannabinoid synthesis andaccumulation abilities, as a small but significantly higherconcentration of cannabinoids (weight per unit area) was found intissues lacking in chlorophyll, demonstrating that plant tissues devoidof chlorophyll could support the synthesis and accumulation ofcannabinoids within their own glandular trichomes, and that the carbonsource required would have been produced elsewhere within the plant andthen translocated to these glandular trichomes, where synthesis andaccumulation of cannabinoids takes place.

Our results suggest that synthesis and accumulation of cannabinoids inglandular trichomes within inflorescences and leaves tissues devoid ofchlorophyll may be inferior than in tissues with higher content ofchlorophyll. We detected an opposite behavior on terpenoid content ininflorescences, suggesting that glandular trichomes within inflorescencetissues devoid of chlorophyll have a similar terpenoid synthesis andaccumulation to glandular trichomes within inflorescence tissues withhigher content of chlorophyll. At the same time, the detected terpenoidcontent in leaves does not support the results obtained ininflorescences, but the different ratios of sessile and capitate stalkedglandular trichomes present in leaves and inflorescence tissues mayexplain the results obtained in this study.

Results were obtained showing that in artificial growing conditions thevariety ‘Pilar’ achieved significantly higher average cannabinoid andterpenoid yields than the variety ‘Divina’. In fact, plants of thevariety ‘Pilar’ achieved a significant (p=0.017) higher average CBDyield per plant (1.79 g of CBD/plant) than the average CBD yield perplant of the variety ‘Divina’ (1.29 g of CBD/plant), as well as asignificant (p=0.00026) higher average Δ9-THC yield per plant (0.18 g ofΔ9-THC/plant) than the average Δ9-THC yield per plant of the variety‘Divina’ (0.13 g of Δ9-THC/plant). In addition, a significant (p=0.0042)higher average in the yield of total terpenoids per plant (0.40 g oftotal terpenoids/plant) was obtained for ‘Pilar’ than for the variety‘Divina’ (0.30 g of total terpenoids/plant). Interestingly, the yieldsobtained on these secondary metabolites by the leaf component werealways higher than the yields obtained from the inflorescences,confirming that both components are of interest for extractingcannabinoids and terpenoids. Also, the average CBD yield obtained perplant of the variety ‘Divina’ (1.29 g of CBD/plant) represents 72% ofthe average CBD yield obtained per plant of the variety ‘Pilar’ (1.79 gof CBD/plant). This primary productive parameter seems to be directlycorrelated with the ratio of chlorophyll contents between ‘Divina’ and‘Pilar’ varieties, which was 73%.

By comparing contents of main terpenoids (expressed as % on w/w basis)determined in inflorescences and leaves of both varieties, slightdifferences in terpenoid composition were detected. In fact, whileslightly higher contents in all the main terpenoids were detected inleaves of the ‘Pilar’ variety, slightly higher contents of theterpenoids α-pinene, β-pinene, terpinolene, and farnesene were detectedin the inflorescences of the ‘Divina’ variety, although no significantdifferences were found in total terpenoid contents in inflorescences.

CONCLUSIONS

Plants of the variety ‘Divina’ cultivated for their esthetic value couldbe eventually harvested and used as an herbal remedy without anypotential risk of abuse, giving a consistent added value to thisdual-purpose variety. Extensive exploitation of an ornamental variegatedCannabis variety as starting material could be potentially consideredfor obtaining essential oil and CBD-enriched extracts with deprivedresidues of chlorophyll, as well as pure forms of CBD, especially whenproduction of vegetable raw material take place in countries withrestrictive rules on exploiting this plant species and where amorphological marker could make a difference on permitting itscultivation.

LITERATURE CITED

-   Casano, S. (2018). Development of ornamental Cannabis sativa L.    varieties: phytochemical, morphological, genetic characterization    and propagation aspects. Acta Horticulturae (under editor review).-   Codesido, V., Marin, M., Sánchez-Carnerero Callado, C.,    Ferreiro-Vera, C., and Casano, S. (2018). Cannabinoids and    terpenoids yields of the ornamental Cannabis sativa L. variety    Divina characterized by a variegated foliage as morphological    marker. Acta Horticulturae (under editor review).-   Lata, H., Chandra, S., Khan, I. A., Elsohly, M. A. (2017).    Micropropagation of Cannabis sativa L. —An Update. In Cannabis    sativa L.—Botany and Biotechnology. S. Chandra, H. Lata, and M. A.    ElSohly, eds. (Oxford, MS: Springer International Publishing), p.    285-297 https://doi.org/10.1007/978-3-319-54564-6_13. PubMed-   Lichtenthaler, H. K. (1987). Chlorophylls and Carotenoids: Pigments    of Photosynthetic Biomembranes. Methods in Enzymology 148, 350-382    https://doi.org/10.1016/0076-6879(87)48036-1. PubMed-   Potter, D. (2009). The propagation, characterisation and    optimisation of Cannabis sativa L. as a phytopharmaceutical. Thesis    for the degree of Doctor of Philosophy (PhD) in Pharmaceutical    Sciences, Department of Pharmaceutical Science Research, King's    College London, March 2009.-   Potter, D. (2013). A review of the cultivation and processing of    Cannabis (Cannabis sativa L.) for production of prescription    medicines in the UK. Drug testing and analysis. Special Issue:    Cannabinoids part II: The current situation with cannabinoids 6,    31-38 https://10.1002/dta.1531. PubMed-   Small, E., and Cronquist, A. (1976). A practical and natural    taxonomy for Cannabis. Taxon 25, 405-435    https://doi.org/10.2307/1220524. PubMed

DETAILED BOTANICAL DESCRIPTION

The following is a detailed botanical description of the new variety‘Divina’. The color codes referred to herein are from the Munsell PlantTissue Color Book and Charts (SKU M50150). These observations on leavesand stem were done on a stock plant having approximately 12 weeks ofage, meaning that since the rooted cutting has been potted, it has beenmaintained for 12 weeks under 18 hours/day of artificial lighting(400-600 μmol m⁻²s⁻¹), and under controlled temperatures and relativehumidity, and CO₂ enriched up to 800 ppm. Observations on inflorescencesand flowers were done on the plant of FIG. 3, the day it was harvested.

-   The plant:    -   -   Type (life form and habitat).—Herbaceous tap-rooted annual.        -   Classification.—Variety of Cannabis sativa L., varietal            denomination ‘Divina’.-   Origin, form and growth characteristics:    -   -   Origin.—Spontaneous natural mutation of the variety Cannabis            sativa L., varietal denomination ‘Pilar’ (CPVO Application            number 2016/0115; granted with decision N° EU 50009 of 16            Jul. 2018).        -   Propagation.—The variety ‘Divina’ is propagated solely by            asexual reproduction, e.g., in vivo rooting of cuttings or            in vitro micropropagation. However, female inflorescences of            plants of the variety ‘Divina’ can produce viable seeds when            pollinated from fertile pollen of other Cannabis varieties.        -   Mature habit.—Tap-rooted annual with an extensive fibrous            root system, upright and much branched aerial portion of the            plant. Natural height is very short and overall size varies            from 0.9-1.1 m tall and 0.3-0.4 m wide across at the widest            point. However, the growth form of all cloned individuals            can be highly manipulated by systematic removal of terminal            buds, inducing a greater branching habit than without            removing terminal buds. The growth form can also be            significatively manipulated in height and width of plants by            changing the period of exposing cloned individuals under a            “long day” artificial light regime (>18 hours/day of light)            and the pedoclimatic conditions of cultivation. Plants do            not flower when exposed to >18 hours/day of light.        -   Growth.—Vigorous annual herb.-   Foliage (leaves):    -   -   Arrangement.—Alternate.        -   Form.—Palmately compound, (3) 5 linear lanceolate leaflets            with glandular hairs.        -   Size.—Whole.        -   Leaf.—11-16 cm long (with petiole), middle (largest) leaflet            7-12 cm long (without petiole), 1.8-2.8 cm wide.        -   Margins.—Coarsely serrate.        -   Color.—Top — variegated green (7.5 GY 3/4) and yellow (2.5            GY 8/4).        -   Color.—Bottom — variegated green (5 GY 5/4) and yellow (2.5            GY 8/2); with lighter colors than in the top of the leaves.        -   Veins.—Bottom — pronounced mid-rib, with straight axial            branches at about a 45° angle, toward distal end of the            leaflet.        -   Color.—Yellow (2.5 GY 8/2).        -   Petiole.—Length 3.0-4.5 cm at maturity.        -   Color.—Yellow (2.5 GY 8/2) in the bottom and reddish (5R            4/6) the top.        -   Stipules.—Present at base of petiole, 0.4-0.7 cm long,            bulbous bases, acuminate (tapering concave to apex).        -   Aroma.—A blend of Citrus fruits and Pinus needles.-   Stem:    -   -   Shape.—Solid, very short internodes, thin, shallow, medium            pith-in cross section, 1,5-2,5 cm diameter at base.        -   Color.—Medium green (2.5 GY 7/8).-   Inflorescence:    -   -   Blooming habit.—Elongated compound cymes or panicles,            generally forming spikes from 0.2 m to 0.4 m in length,            densely packed with individual small pistillate flowers            subtended by small leaves, these with densely packed            capitate glandular trichomes.        -   Aroma.—A blend of Citrus fruits and Pinus needles.-   Flowers:    -   -   Corolla.—Petals and calyx unified and collectively appressed            to and surrounding the ovary.        -   Color.—Variegated green (7.5 GY 3/4) and yellow (2.5 GY            8/4).        -   Diameter.—Apical cyme has 4-6 cm diameter.        -   Shape.—Elliptical.        -   Involucral bracts.—Present, several glandular trichomes            present on its surface, enclose the flower.        -   Calyces.—Appressed to the base of the ovary with the corolla            as a unified perianth.        -   Color.—Variegated green (7.5 GY 3/4) and yellow (2.5 GY            8/4).        -   Filaments.—N/A, no staminate flowers observed.        -   Stigma.—Length 5-7 mm, about 1 mm wide at base, tapering to            distal end. Densely covered with minute (<1 mm) hairs.        -   Color.—Lemony white (2.5 GY 8/2), drying slowly to orange            (2.5 YR 5/8) from apex to base after anthesis.        -   Number.—2.        -   Staminate column.—N/A, no staminate flowers observed.        -   Fruit.—An achene in this genus; however, no fruits were seen            in absence of viable pollen while fruits were seen in            presence of viable pollen produced by other monoecious or            dioecious male plants.        -   Pollen.—N/A, no staminate flowers observed; however, the            artificial production of staminate flowers can be induced by            applying silver thiosulphate (STS) to plants in vegetative            stage or at the beginning of the flowering stage, meaning            that the induced production of viable pollen can be            artificially obtained.        -   Petalage.—The plant is essentially without petals            (apetalous); these are fused and appressed to the base of            the ovary with the calyx as the perianth.        -   Pedicel.—Flowers are essentially sessile (attached to the            stem), and as such have no pedicel.-   General characteristics and culture:    -   -   Blooming period.—Plants will bloom in 4-6 weeks when 12            hours of light/day are applied to induce flowering.        -   Hardiness.—Probably it is fairly hardy; however, hardiness            in nature is unknown as this plant has only been cultivated            under controlled conditions.        -   Breaking action.—Stems are fibrous, strong and flexible,            highly resistant to breakage.        -   Rooting.—Approximately 70% success rate in vitro rooting            with cuttings. After exposing for 2 minutes the basal part            of the cuttings to a natural rooting product called            “Inabar”, treated cuttings were placed in Jiffy cubes (model            7; 44 mm) in propagation. The ranges adopted for climatic            parameters were: 22±1° C., 75-85% of relative humidity,            350-400 ppm of carbon dioxide, and under a photon flux            density of approximately 100 μE mol m⁻²s⁻¹ provided by            light-emitting diodes (LEDs) lights under continuous            daylength (24 hours light/day). ‘Divina’ is susceptible to            fungal disease during in vitro rooting.        -   Growth regulator.—No growth regulators or hormones were used            in the cultivation stages, while in the propagation stage a            liquid rooting product containing natural phytohormones was            used for stimulating the formation of root primordia and            their elongation.        -   Shipping tolerance.—The plant has been successfully shipped            at controlled temperature (20° C.) by using Clone Shipper,            which is a closed shipping container provided by a LED light            to keep the plant in the vegetative stage while being            shipped for 3-4 days before arriving to its destination.

It is claimed:
 1. A new and distinct variety of a Cannabis sativa L.plant, as shown and described, having variegated yellow and greenfoliage.